YAP and the RhoC regulator ARHGAP18, are required to mediate flow-dependent endothelial cell alignment

BACKGROUND: Vascular endothelial cell alignment in the direction of flow is an adaptive response that protects against aortic diseases such as atherosclerosis. The RhoGTPases are known to regulate this alignment. We have shown previously that ARHGAP18 in endothelial cells is a negative regulator of RhoC and its expression is essential in flow-mediated alignment. Depletion of ARHGAP18 inhibits alignment and results in the induction of a pro-inflammatory phenotype. In embryogenesis, ARHGAP18 was identified as a downstream effector of the Yes-associated protein, YAP, which regulates cell shape and size. METHODS: We have used siRNA technology to deplete either ARHGAP18 or YAP in human endothelial cells. The in vitro studies were performed under athero-protective, laminar flow conditions. The analysis of YAP activity was also investigated, using high performance confocal imaging, in our ARHGAP18 knockout mutant mice. RESULTS: We show here that loss of ARHGAP18, although decreasing the expression of YAP results in its nuclear localisation consistent with activation. We further show that depletion of YAP itself results in its activation as defined by an in increase in its nuclear localisation and an increase in the YAP target gene, CyR61. Depletion of YAP, similar to that observed for ARHGAP18 depletion, results in loss of endothelial cell alignment under high shear stress mediated flow and also in the activation of NFkB, as determined by p65 nuclear localisation. In contrast, ARHGAP18 overexpression results in upregulation of YAP, its phosphorylation, and a decrease in the YAP target gene Cyr61, consistent with YAP inactivation. Finally, in ARHGAP18 deleted mice, in regions where there is a loss of endothelial cell alignment, a situation associated with a priming of the cells to a pro-inflammatory phenotype, YAP shows nuclear localisation. CONCLUSION: Our results show that YAP is downstream of ARHGAP18 in mature endothelial cells and that this pathway is involved in the athero-protective alignment of endothelial cells under laminar shear stress. ARHGAP18 depletion leads to a disruption of the junctions as seen by loss of VE-Cadherin localisation to these regions and a concomitant localisation of YAP to the nucleus.